Rodrigo I. Santos

Spinal Cord Ventral Horns and Lymphoid Organ Involvement in Powassan Virus Infection in a Mouse Model

Powassan virus (POWV) belongs to the family Flaviviridae and is a member of the tick-borne encephalitis serogroup. Transmission of POWV from infected ticks to humans has been documented in the USA, Canada, and Russia, causing fatal encephalitis in 10% of human cases and significant neurological sequelae in survivors. We used C57BL/6 mice to investigate POWV infection and pathogenesis. After footpad inoculation, infected animals exhibited rapid disease progression and 100% mortality. Immunohistochemistry and immunofluorescence revealed a very strong neuronal tropism of POWV infection. The central nervous system infection appeared as a meningoencephalitis with perivascular mononuclear infiltration and microglial activation in the brain, and a poliomyelitis-like syndrome with high level of POWV antigen at the ventral horn of the spinal cord. Pathological studies also revealed substantial infection of splenic macrophages by POWV, which suggests that the spleen plays a more important role in pathogenesis than previously realized. This report provides a detailed description of the neuroanatomical distribution of the lesions produced by POWV infection in C57BL/6 mice.

The Ebola Interferon Inhibiting Domains Attenuate and Dysregulate Cell-Mediated Immune Responses

Ebola virus (EBOV) infections are characterized by deficient T-lymphocyte responses, T-lymphocyte apoptosis and lymphopenia. We previously showed that disabling of interferon-inhibiting domains (IIDs) in the VP24 and VP35 proteins effectively unblocks maturation of dendritic cells (DCs) and increases the secretion of cytokines and chemokines. Here, we investigated the role of IIDs in adaptive and innate cell-mediated responses using recombinant viruses carrying point mutations, which disabled IIDs in VP24 (EBOV/VP24m), VP35 (EBOV/VP35m) or both (EBOV/VP35m/VP24m). Peripheral blood mononuclear cells (PBMCs) from cytomegalovirus (CMV)-seropositive donors were inoculated with the panel of viruses and stimulated with CMV pp65 peptides. Disabling of the VP35 IID resulted in increased proliferation and higher percentages of CD4+ T cells secreting IFNγ and/or TNFα. To address the role of aberrant DC maturation in the IID-mediated suppression of T cell responses, CMV-stimulated DCs were infected with the panel of viruses and co-cultured with autologous T-lymphocytes. Infection with EBOV/VP35m infection resulted in a significant increase, as compared to wt EBOV, in proliferating CD4+ cells secreting IFNγ, TNFα and IL-2. Experiments with expanded CMV-specific T cells demonstrated their increased activation following co-cultivation with CMV-pulsed DCs pre-infected with EBOV/VP24m, EBOV/VP35m and EBOV/VP35m/VP24m, as compared to wt EBOV. Both IIDs were found to block phosphorylation of TCR complex-associated adaptors and downstream signaling molecules. Next, we examined the effects of IIDs on the function of B cells in infected PBMC. Infection with EBOV/VP35m and EBOV/VP35m/VP24m resulted in significant increases in the percentages of phenotypically distinct B-cell subsets and plasma cells, as compared to wt EBOV, suggesting inhibition of B cell function and differentiation by VP35 IID. Finally, infection with EBOV/VP35m increased activation of NK cells, as compared to wt EBOV. These results demonstrate a global suppression of cell-mediated responses by EBOV IIDs and identify the role of DCs in suppression of T-cell responses.

Immune cell targets of infection at the tick-skin interface during powassan virus transmission

Powassan virus (POWV) is a tick-borne flavivirus that can result in a severe neuroinvasive disease with 50% of survivors displaying long-term neurological sequelae. Human POWV cases have been documented in Canada, the United States, and Russia. Although the number of reported POWV human cases has increased in the past fifteen years, POWV remains one of the less studied human pathogenic flaviviruses. Ixodes ticks are the vectors for POWV, and the virus is transmitted to a host’s skin very early during the tick feeding process. Central to the successful transmission of a tick-borne pathogen are complex interactions between the host immune response and early tick-mediated immunomodulation, all of which initially occur at the skin interface. In our prior work, we examined the cutaneous immune gene expression during the early stages of POWV-infected Ixodes scapularis feeding. The present study serves to further investigate the skin interface by identifying early cell targets of infection at the POWV-infected tick feeding site. An in vivo infection model consisting of POWV-infected ticks feeding on mice for short durations was used in this study. Skin biopsies from the tick feeding sites were harvested at various early time points, enabling us to examine the skin histopathology and detect POWV viral antigen in immune cells present at the tick feeding site. The histopathology from the present study demonstrates that neutrophil and mononuclear cell infiltrates are recruited earlier to the feeding site of a POWV-infected tick versus an uninfected tick. This is the first report demonstrating that macrophages and fibroblasts contain POWV antigens, which suggests that they are early cellular targets of infection at the tick feeding site. These data provide key insights towards defining the complex interactions between the host immune response and early tick-mediated immunomodulation.

Ebola virus glycoprotein directly triggers T lymphocyte death despite of the lack of infection

Fatal outcomes of Ebola virus (EBOV) infections are typically preceded by a ‘sepsis-like’ syndrome and lymphopenia despite T cells being resistant to Ebola infection. The mechanisms that lead to T lymphocytes death remain largely unknown; however, the degree of lymphopenia is highly correlative with fatalities. Here we investigated whether the addition of EBOV or its envelope glycoprotein (GP) to isolated primary human CD4+ T cells induced cell death. We observed a significant decrease in cell viability in a GP-dependent manner, which is suggestive of a direct role of GP in T cell death. Using immunoprecipitation assays and flow cytometry, we demonstrate that EBOV directly binds to CD4+ T cells through interaction of GP with TLR4. Transcriptome analysis revealed that the addition of EBOV to CD4+ T cells results in the significant upregulation of pathways associated with interferon signaling, pattern recognition receptors and intracellular activation of NFκB signaling pathway. Both transcriptome analysis and specific inhibitors allowed identification of apoptosis and necrosis as mechanisms associated with the observed T cell death following exposure to EBOV. The addition of the TLR4 inhibitor CLI-095 significantly reduced CD4+ T cell death induced by GP. EBOV stimulation of primary CD4+ T cells resulted in a significant increase in secreted TNFα; inhibition of TNFα-mediated signaling events significantly reduced T cell death while inhibitors of both necrosis and apoptosis similarly reduced EBOV-induced T cell death. Lastly, we show that stimulation with EBOV or GP augments monocyte maturation as determined by an overall increase in expression levels of markers of differentiation. Subsequently, the increased rates of cellular differentiation resulted in higher rates of infection further contributing to T cell death. These results demonstrate that GP directly subverts the host’s immune response by increasing the susceptibility of monocytes to EBOV infection and triggering lymphopenia through direct and indirect mechanisms.

Spread of Oropouche Virus into the Central Nervous System in Mouse

Oropouche virus (OROV) is an important cause of arboviral illness in Brazil and other Latin American countries, with most cases clinically manifested as acute febrile illness referred to as Oropouche fever, including myalgia, headache, arthralgia and malaise. However, OROV can also affect the central nervous system (CNS) with clinical neurological implications. Little is known regarding OROV pathogenesis, especially how OROV gains access to the CNS. In the present study, neonatal BALB/c mice were inoculated with OROV by the subcutaneous route and the progression of OROV spread into the CNS was evaluated. Immunohistochemistry revealed that OROV infection advances from posterior parts of the brain, including the periaqueductal gray, toward the forebrain. In the early phases of the infection OROV gains access to neural routes, reaching the spinal cord and ascending to the brain through brainstem regions, with little inflammation. Later, as infection progresses, OROV crosses the blood-brain barrier, resulting in more intense spread into the brain parenchyma, with more severe manifestations of encephalitis.

Ebola virus binding to Tim-1 on T lymphocytes induces a cytokine storm

ABSTRACT Ebola virus (EBOV) disease (EVD) results from an exacerbated immunological response that is highlighted by a burst in the production of inflammatory mediators known as a “cytokine storm.” Previous reports have suggested that nonspecific activation of T lymphocytes may play a central role in this phenomenon. T-cell immunoglobulin and mucin domain-containing protein 1 (Tim-1) has recently been shown to interact with virion-associated phosphatidylserine to promote infection. Here, we demonstrate the central role of Tim-1 in EBOV pathogenesis, as Tim-1−/− mice exhibited increased survival rates and reduced disease severity; surprisingly, only a limited decrease in viremia was detected. Tim-1−/− mice exhibited a modified inflammatory response as evidenced by changes in serum cytokines and activation of T helper subsets. A series of in vitro assays based on the Tim-1 expression profile on T cells demonstrated that despite the apparent absence of detectable viral replication in T lymphocytes, EBOV directly binds to isolated T lymphocytes in a phosphatidylserine–Tim-1-dependent manner. Exposure to EBOV resulted in the rapid development of a CD4Hi CD3Low population, non-antigen-specific activation, and cytokine production. Transcriptome and Western blot analysis of EBOV-stimulated CD4+ T cells confirmed the induction of the Tim-1 signaling pathway. Furthermore, comparative analysis of transcriptome data and cytokine/chemokine analysis of supernatants highlight the similarities associated with EBOV-stimulated T cells and the onset of a cytokine storm. Flow cytometry revealed virtually exclusive binding and activation of central memory CD4+ T cells. These findings provide evidence for the role of Tim-1 in the induction of a cytokine storm phenomenon and the pathogenesis of EVD. IMPORTANCE Ebola virus infection is characterized by a massive release of inflammatory mediators, which has come to be known as a cytokine storm. The severity of the cytokine storm is consistently linked with fatal disease outcome. Previous findings have demonstrated that specific T-cell subsets are key contributors to the onset of a cytokine storm. In this study, we investigated the role of Tim-1, a T-cell-receptor-independent trigger of T-cell activation. We first demonstrated that Tim-1-knockout (KO) mice survive lethal Ebola virus challenge. We then used a series of in vitro assays to demonstrate that Ebola virus directly binds primary T cells in a Tim-1–phosphatidylserine-dependent manner. We noted that binding induces a cytokine storm-like phenomenon and that blocking Tim-1–phosphatidylserine interactions reduces viral binding, T-cell activation, and cytokine production. These findings highlight a previously unknown role of Tim-1 in the development of a cytokine storm and “immune paralysis.” IMPORTANCE Ebola virus infection is characterized by a massive release of inflammatory mediators, which has come to be known as a cytokine storm. The severity of the cytokine storm is consistently linked with fatal disease outcome. Previous findings have demonstrated that specific T-cell subsets are key contributors to the onset of a cytokine storm. In this study, we investigated the role of Tim-1, a T-cell-receptor-independent trigger of T-cell activation. We first demonstrated that Tim-1-knockout (KO) mice survive lethal Ebola virus challenge. We then used a series of in vitro assays to demonstrate that Ebola virus directly binds primary T cells in a Tim-1–phosphatidylserine-dependent manner. We noted that binding induces a cytokine storm-like phenomenon and that blocking Tim-1–phosphatidylserine interactions reduces viral binding, T-cell activation, and cytokine production. These findings highlight a previously unknown role of Tim-1 in the development of a cytokine storm and “immune paralysis.”

Detection of Rickettsia amblyommii in ticks collected from Missouri, USA.

Dear Editor, In September 2012, we collected ticks from Missouri with the goal of isolating the novel phlebovirus, Heartland virus (HRTV). HRTV was described in two farmers from northwestern Missouri who presented with thrombocytopenia and severe febrile illness.1 These patients were both bitten by ticks 5–7 days before the onset of their clinical symptoms. Our hypothesis was that we would isolate HRTV from ticks collected in Missouri by inoculation of cell culture and/or by detection of viral RNA on polymerase chain reaction (PCR) assay. Furthermore, we used this field surveillance study as an opportunity to screen for other potential viral and bacterial pathogens in the tick samples we collected. Information from the published literature1 was used to identify three geographically relevant collection sites across the central and western region of the state (Supplementary Figure S1A). One thousand two hundred and sixty-nine total ticks were collected from the three locations. Of these, 1191 (93.9%) were Dermacentor albipictus, 74 (5.8%) were Amblyomma americanum and four (0.3%) were Ixodes scapularis (Supplementary Figure S1B). Two nymphs were collected at location 2, but all other ticks collected during this study were larvae. The speciated ticks were pooled into groups of twenty and screened for tick-borne pathogens (Supplementary Methods and Supplementary Table S1). Using the primers specific for HRTV,1 Powassan virus2 and deer tick virus,3 we were unable to generate any positive PCR amplicons in the viral PCR screening. At location 2, one larval pool and one nymphal pool of ticks generated positive PCR amplicons when screened with the Rickettsia-specific primers for the outer membrane protein A (ompA) and citrate synthase (gltA) genes.4 Sequence analysis demonstrated that the two Rickettisa-positive samples aligned with the ompA and gltA genes of Candidatus Rickettsia amblyommii [GenBank: 378930552]. ompA gene sequences for R. amblyommii, R. raoultii, R. slovaca and R. rickettsii were obtained from GenBank. These sequences were trimmed and then underwent ClustalW alignment in the MegAlign program. Specifically, ompA-positive samples from both the larval and nymphal pools shared 100% sequence identity across a 431 bp segment of the ompA gene of Candidatus Rickettsia amblyommii [GenBank: 378930552] (Figure 1A). Additional sequence analysis demonstrated that there was 100% sequence identity between both the larval and nymphal gltA-positive samples and Candidatus Rickettsia amblyommii [GenBank: 378930552] across a 628 bp segment of the gltA gene (Supplementary Figure S2). Furthermore, the presence of Rickettsia in a larval pool of ticks collected at location 2 indicates the occurrence of transovarial transmission of R. amblyommii. Figure 1 Molecular detection of R. amblyommii. (A) Multiple sequence alignment of the outer membrance protein A (ompA) gene. Sequence ruler applies to R. amblyommii omp A sequence. (B) R. amblyommii polyclonal antibody-stained tick homogenate. Tick homogenates ... To further confirm our molecular identification of R. amblyommii, we screened the tick samples with Rickettsia-specific primers for the outer membrane protein B (ompB) gene5 and for the 17 kDa gene.6 The tick samples aligned perfectly with the Candidatus Rickettsia amblyommii [GenBank: 378930552] ompB gene sequence (Supplementary Figure S3). Our R. amblyommii-positive samples also completely aligned with the Candidatus Rickettsia amblyommii [GenBank: 378930552] 17 kDa gene sequence (Supplementary Figure S4). No cytopathic effect was detected in any of the cell lines inoculated with the tick homogenates. However, we used an immunofluorescence assay (Supplementary Methods) to confirm the detection of R. amblyommii antigens in the A. americanum tick homogenates (Figure 1B). Tick mitochondrial 16S rRNA sequence analysis confirmed that the R. amblyommii-positive samples were isolated from A. americanum ticks. The 16S rRNA sequence from our Rickettsia-positive tick pools shared 100% sequence identity with A. americanum 16S rRNA (Supplementary Figure S5). This field surveillance study was unable to isolate HRTV in any of the ticks we collected from Missouri. The lack of HRTV found in this study may be the result of our small sample sizes; only 5.8% of the total collected ticks were A. americanum. After our field collection of ticks was completed, another group published their findings of detecting HRTV from A. americanum collected in Missouri during 2012.7 This group conducted tick collections ranging from April to early-August 2012. As our collection did not occur until mid-September, the majority of ticks collected in our study were larvae, as would be expected.8 We did confirm the presence of R. amblyommii in two pooled A. americanum tick homogenates. To date, no definitive role has been defined for R. amblyommii in human pathogenesis, but a recent study has shown that A. americanum ticks parasitizing humans are frequently infected with R. amblyommii.9 Two A. americanum ticks collected in Kansas were found to be concurrently infected with R. rickettsii, which causes Rocky Mountain spotted fever, and with R. amblyommii.10 The co-infection of these A. americanum ticks with R. rickettsii and R. amblyommii raises interesting questions about the epidemiology of spotted fever group rickettsiae and Rocky Mountain spotted fever. A recent study in North Carolina screened ticks for spotted fever group rickettsiae and found that there was a high prevalence of A. americanum ticks infected with R. amblyommii.11 As this tick species is relatively aggressive and readily parasitizes humans, the authors suggested that some cases of rickettsiosis diagnosed as Rocky Mountain spotted fever in North Carolina may instead be caused by R. amblyommii. Because other Rickettsia species, such as R. parkeri, were initially thought to be endosymbionts but were later shown to be pathogenic, it is important to continue evaluating the potential public health threat that R. amblyommii-infected A. americanum ticks pose to the humans they parasitize.

Transcriptional immunoprofiling at the Tick-Virus-Host interface during early stages of tick-borne encephalitis virus transmission

Emerging and re-emerging diseases transmitted by blood feeding arthropods are significant global public health problems. Ticks transmit the greatest variety of pathogenic microorganisms of any blood feeding arthropod. Infectious agents transmitted by ticks are delivered to the vertebrate host together with saliva at the bite site. Tick salivary glands produce complex cocktails of bioactive molecules that facilitate blood feeding and pathogen transmission by modulating host hemostasis, pain/itch responses, wound healing, and both innate and adaptive immunity. In this study, we utilized Illumina Next Generation Sequencing to characterize the transcriptional immunoprofile of cutaneous immune responses to Ixodes ricinus transmitted tick-borne encephalitis virus (TBEV). A comparative immune gene expression analysis of TBEV-infected and uninfected tick feeding sites was performed. Our analysis reveals that ticks create an inflammatory environment at the bite site during the first 3 h of feeding, and significant differences in host responses were observed between TBEV-infected and uninfected tick feeding. Gene-expression analysis reveals modulation of inflammatory genes after 1 and 3 h of TBEV-infected tick feeding. Transcriptional levels of genes specific to chemokines and cytokines indicated a neutrophil-dominated immune response. Immunohistochemistry of the tick feeding site revealed that mononuclear phagocytes and fibroblasts are the primary target cells for TBEV infection and did not detect TBEV antigens in neutrophils. Together, the transcriptional and immunohistochemistry results suggest that early cutaneous host responses to TBEV-infected tick feeding are more inflammatory than expected and highlight the importance of inflammatory chemokine and cytokine pathways in tick-borne flavivirus transmission.

Role of Transmembrane Protein 16F in the Incorporation of Phosphatidylserine Into Budding Ebola Virus Virions

Viral apoptotic mimicry, which is defined by exposure of phosphatidylserine (PtdSer) into the outer leaflet of budding enveloped viruses, increases viral tropism, infectivity and promotes immune evasion. Here, we report that the calcium (Ca2+)-dependent scramblase, transmembrane protein 16F (TMEM16F), is responsible for the incorporation of PtdSer into virion membranes during Ebola virus infection. Infection of Huh7 cells with Ebola virus resulted in a pronounced increase in plasma membrane-associated PtdSer, which was demonstrated to be dependent on TMEM16F function. Analysis of virions using imaging flow cytometry revealed that short hairpin RNA-mediated down-regulation of TMEM16F function directly reduced virion-associated PtdSer. Taken together, these studies demonstrate that TMEM16F is a central cellular factor in the exposure of PtdSer in the outer leaflet of viral membranes.

Disruption of Phosphatidylserine Synthesis or Trafficking Reduces Infectivity of Ebola Virus

The outer leaflet of the viral membrane of Ebola virus (EBOV) virions is enriched with phosphatidylserine (PtdSer), which is thought to play a central role in viral tropism, entry, and virus-associated immune evasion. We investigated the effects of inhibiting synthesis and/or export of PtdSer to the cell surface of infected cells on viral infectivity. Knockdown of both PtdSer synthase enzymes, PTDSS1 and PTDSS2, effectively decreased viral production. Decreased PtdSer expression resulted in an accumulation of virions at the plasma membrane and adjacent of intracellular organelles, suggesting that virion budding is impaired. The addition of inhibitors that block normal cellular trafficking of PtdSer to the plasma membrane resulted in a similar accumulation of virions and reduced viral replication. These findings demonstrate that plasma membrane-associated PtdSer is required for efficient EBOV budding, increasing EBOV infectivity, and could constitute a potential therapeutic target for the development of future countermeasures against EBOV.